Engineered Protein Nanocarriers for Intracellular Antibody Delivery There is a long list of ?undruggable? targets, disease related protein-protein interactions inside cells that small molecule drugs cannot block. Antibodies, in particular, can be engineered to bind almost any protein and inhibit protein function but have not yet been used against these targets. This is primarily due to the significant, unmet challenge in delivering sufficient amounts of folded, functional antibodies inside cells. The goal of this proposal is to build an intracellular antibody carrier using protein self-assembly and assess the therapeutic activity of delivered antibodies. By utilizing a hexameric protein bundle (HEX) and generic antibody-binding peptides (SPABs), nanoscale complexes containing only protein can be formed with antibodies. Our hypothesis is that the HEX antibody nanocarrier will significantly increase intracellular antibody uptake compared to soluble antibodies, and will enable therapeutic antibody binding to cytosolic targets. Our preliminary data suggests that Hex-SPAB protein nanocarriers do deliver functional antibody into the cytosol and, therefore, could be broadly applicable to future intracellular antibody therapeutics. Three aims have been set to meet the objective and test the hypothesis. (1) Fabricate and characterize the size, stability and antibody loading of HEX antibody nanocarriers. (2) Measure cellular uptake, cytosolic localization, and therapeutic function of model anti-cancer antibodies delivered by HEX nanocarriers. (3) Determine the therapeutic potential of HEX nanocarriers by adding anti-HER2 targeting antibodies to direct carriers to breast cancer cells, and evaluate the blood circulation time and immunogenicity of HEX nanocarriers in vivo. Two main outcomes are expected. First, an innovative protein nanocarrier will be created that facilitates cytosolic delivery of functional therapeutic antibodies. It will provide proof of concept for intracellular antibody therapeutics to inhibit proteins, including combinations with targeting antibodies. Second, this work will measure key properties necessary for translation, blood circulation time and immunogenicity. This will open the door for intracellular antibody engineering comparable to current extracellular antibodies that are successfully treating human disease.